CN104418950A - Human asialoglycoprotein receptor (ASGPR) monoclonal antibody as well as preparation method and application thereof - Google Patents
Human asialoglycoprotein receptor (ASGPR) monoclonal antibody as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a human asialoglycoprotein receptor (ASGPR) monoclonal antibody as well as a preparation method and application thereof. The monoclonal antibody can be used for well recognizing an ASGPR antigen, cannot perform cross reaction with other proteins, and is very high in specificity and sensitivity. The antibody disclosed by the invention can be applied to identification of primary hepatocellular carcinoma and metastatic liver cancer, separation and detection of circulating liver cancer cells, liver imaging and targeted delivery of liver medicines or genes.
Description
Technical field
The invention belongs to biological technical field, relate to one and to identify specifically and in conjunction with the monoclonal antibody of people's asialoglycoprotein receptor (Asialoglycoprotein Receptor, ASGPR) extracellular fragment, its preparation method and application thereof.
Background technology
Asialoglycoprotein receptor (asialoglycoprotein receptor, ASGPR), also known as hepatic lectin or glycogen aggegation albumen, specific expressed in Mammals hepatic sinusoid and basolateral hepatic parenchymal cells surface, main Physiological Function is remove the glycoprotein, lipoprotein and the apoptotic cell that contain D-galactose residue or N-acetylgalactosamine residue in Peripheral Circulation.The liver cell specificity of the physiological function of ASGPR uniqueness and height thereof, makes to have important fundamental research meaning and potential clinical value in the targeted delivery etc. of its discriminating at hepatocellular carcinoma and secondary liver cancer, the separation and detection of circulation liver cancer cell, liver imaging and liver drug or gene.
Existing people utilizes ASGPR1 full-length gene [Trahtenherts A in recent years, Benhar I.Aninternalizing antibody specific for the human asialoglycoprotein receptor.Hybridoma (Larchmt), 2009, 28:225-233.], the large subunit isomer protein H1b of ASGPR [Liu Jia, Ding Honghui, Yang Yan, Hu Bin, Yu Yuan, Huang Hongping, Deng. the Preparation and identification of anti-human ASGPR large subunit isomer protein H1b polyclonal antibody. cell and molecular immunology magazine, 2009, 10:917-919.], ASGPR large subunit sugar cog region gene [Zhao X, Yu Z, Dai W, Yao Z, Zhou w, Cao L, etal.Construction and functional characterization of an anti-asialoglycoproteinreceptor single-chain variable-fragment-targeted melittin.Biotechnol ApplBiochem, 2011, 58:405-411.], ASGPR gene fragment [Park JH, Kim KL, Cho EW.Detection of surface asialoglycoprotein receptor in hepatic and extra-hepaticcells using a novel monoclonal antibody.Biotechnol Lett, 2006, 28:1061-1069.] etc. preparation ASGPR antibody.Regrettably, just can commercially available [santa cruz biotech firm of the U.S. in the monoclonal antibody having Santa company in the recent period only, http://www.scbt.com/table-asgpr.html], and only have a mouse monoclonal antibody for ASGPR1 full length sequence [sc-52623].But, the rarely seen report of the applied research about these antibody.And the antibody restricted application of Santa sold, the needs of clinical application cannot be met.
Therefore, this area is necessary to develop the good ASGPR monoclonal antibody of specificity further, to realizing the clinical conversion of ASGPR using value.
Summary of the invention
The object of the present invention is to provide people's asialoglycoprotein receptor monoclonal antibody and Synthesis and applications thereof.
In a first aspect of the present invention, provide a kind of monoclonal antibody, it is the monoclonal antibody that the hybridoma cell strain being CCTCC NO:C201370 by preserving number is secreted.
In a preference, described monoclonal antibody is Immunoglobulin Isotype IgG1 type.
In another preference, described monoclonal antibody prepares with the antigen-immunized animal shown in SEQ ID NO:1.
In another aspect of this invention, provide a kind of hybridoma cell strain, it is CCTCC NO:C201370 at the preserving number of China typical culture collection center.
In another aspect of this invention, provide a kind of immune conjugate, described immune conjugate comprises: described monoclonal antibody and the marker be attached thereto; Described marker is selected from (but being not limited to): horseradish peroxidase, alkaline phosphatase, vitamin H or fluorescein.
In another aspect of this invention, provide a kind of detection kit, comprising:
Described monoclonal antibody; Or
Described hybridoma cell strain; Or
Described immune conjugate.
In a preference, also comprise in described test kit:
Second antibody, the described monoclonal antibody described in second antibody specific recognition;
Immunomagnetic beads;
Developer;
Washings; And/or
Stop buffer.
In another aspect of this invention, provide the purposes of described monoclonal antibody, for the preparation of detection reagent or the test kit of specific detection people asialoglycoprotein receptor.
In a preference, described detection people asialoglycoprotein receptor comprises: (1) qualitative detection people asialoglycoprotein receptor; Or (2) detection by quantitative people asialoglycoprotein receptor.
In another preference, described qualitative detection comprises: by the expression of the method identifier asialoglycoprotein receptor of western blotting method or immunofluorescence.
In another preference, described detection by quantitative comprises: carried out quantitatively the intensity of cell expressing people asialoglycoprotein receptor or positive rate by flow cytometry, or is carried out quantitatively the expression of people's asialoglycoprotein receptor by double antibody sandwich ELISA.
In another preference, described detection reagent or test kit also for:
The discriminating of hepatocellular carcinoma and secondary liver cancer;
The separation and detection of circulation liver cancer cell;
Liver imaging; With
The targeted delivery of liver drug or gene.
In another aspect of this invention, a kind of separation method of the liver cancer cell that circulates is provided, comprises the steps:
A () is separated mononuclearcell (wherein containing circulation liver cancer cell) from the vitro samples containing circulation liver cancer cell;
B () mixes with mononuclearcell with first antibody, thus form " liver cancer cell-first antibody " binary complex; Described first antibody is described monoclonal antibody;
C () has the magnetic bead of second antibody to mix with described binary complex with coupling, thus form " liver cancer cell-first antibody-second antibody-magnetic bead " tetraplex; With
Tetraplex in (d) magnetic separation step (c).
In a preference, the separation method that described separation mononuclearcell is taked is density gradient cytopheresis.
In another preference, described sample is selected from marrow, blood or other body fluid; Preferably, other body fluid described are lymph liquid, hydrothorax or ascites.
Other aspects of the present invention, due to disclosure herein, are apparent to those skilled in the art.
Accompanying drawing explanation
Fig. 1, Flow cytometry tumor cell line express ASGPR.
The situation that Fig. 2, immunohistochemical method detect normal liver tissue (A), liver cancer tissue (B), intestinal cancer hepatic metastases tissue (C) express ASGPR.
Fig. 3, immunohistofluorescence stain method detect liver cancer tissue and express ASGPR.
Fig. 4, immunocytochemistry detect hepatocellular carcinoma H22 (positive) and breast cancer cell MCF-7 (feminine gender) expresses ASGPR.
Fig. 5, immunocyte fluorescent staining method detect hepatocellular carcinoma H22 (positive) and breast cancer cell MCF-7 (feminine gender) expresses ASGPR.
Fig. 6, immunocyte fluorescent staining method qualification isolated circulation liver cancer cell from Peripheral Blood of Patients with Hepatocellular Carcinoma, wherein Hep Par1+/CD45-/DAPI+ is circulation liver cancer cell, and Hep Par1-/CD45+/DAPI+ is mononuclearcell.
Embodiment
The present inventor, through studying widely, provides a kind of monoclonal antibody of specific anti-human asialoglycoprotein receptor (ASGPR).Described monoclonal antibody can identify ASGPR antigen well, and not with other albumen generation cross reactions, specificity and sensitivity are all very high.Can be applicable to the targeted delivery of the discriminating of hepatocellular carcinoma and secondary liver cancer, the separation and detection of circulation liver cancer cell, liver imaging and liver drug or gene.
Monoclonal antibody
Those skilled in the art all understand, a kind of antigen may contain multiple epitope (antigenic determinant), therefore, can obtain more than antibody for same antigen, the binding characteristic (as specificity etc.) of these antibody to antigen may be all different.Therefore, for same antigen, those skilled in the art need to carry out a large amount of repeatedly to compare, screening and identification, just can find the monoclonal antibody of specific binding target antigen.Because ASGPR aminoacid sequence is long, a lot of antigenic determinant is enclosed in the inside of protein structure, is therefore difficult to the monoclonal antibody finding a species specificity high.When some anti-ASGPR monoclonal antibodies are used to actual detection, suffer from the problem with natural A SGPR protein binding characteristics difference unavoidably, this may be because in testing sample, the antigenic determinant of ASGPR albumen is enclosed in protein structure inside, cannot touch that antibody causes.
For above-mentioned technical barrier, the fragment that the present inventor have chosen multiple ASGPR albumen carrys out immune animal, obtains the splenocyte of animal for the preparation of hybridoma cell strain, thus finds the suitable fragment for immunity.After a large amount of screening, the present inventor finds, the one section of aminoacid sequence (SEQ ID NO:1) come from ASGPR aminoacid sequence 61-291aa position has good immunogenicity, further across mono-clonal screening and checking, thus obtains monoclonal antibody of the present invention.ASGPR is expressed on cytolemma, and the region of monoclonal antibody identification of the present invention just ASGPR be positioned at the epi-position on cell surface, and its trans-membrane region of non-identifying or film inner compartment, therefore monoclonal antibody of the present invention is particularly suitable for the qualification carrying out ASGPR.
Monoclonal antibody of the present invention has very high specificity for ASGPR albumen, is not incorporated into other albumen beyond ASGPR.Further, when for detecting, accurate detected result can be obtained without the need to carrying out too much process to testing sample.Further, based on described monoclonal antibody, the test kit detecting ASGPR exactly can be prepared.
Monoclonal antibody of the present invention utilizes hybridoma technology to prepare (see Kohler etc., Nature256:495,1975; The people such as Kohler, Eur.J.Immunol.6:511,1976; Kohler etc., Eur.J.Immunol.6:292,1976; Hammerling etc., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).A kind of preparation method of hybridoma is:
(1) mouse immune: select of the right age mouse, carries out immunity with antigen peptide to mouse;
(2) cultivation of murine myeloma cell: cultivate murine myeloma cell SP2/0 and make it the good growth conditions of maintenance for cytogamy;
(3) cytogamy: using mouse boosting cell as feeder cell.By the sacrifice got ready in (1), prepare immune spleen cell suspension.Collect the murine myeloma cell in (2).By centrifugal for above-mentioned two kinds of cytomixis, then merge with polyoxyethylene glycol (PEG) mediated cell.Cell after fusion suitably dilutes, and is seeded to culture plate, and felicity condition is cultivated;
(4) screening of hybridoma: above-mentioned culture is cultivated in selective medium.When cell colony grows to size to fit, draw nutrient solution supernatant and do Identification of the antibodies, screening positive clone;
(5) cloning of hybridoma: with limiting dilution assay cloned hybridoma cell, is seeded to 96 orifice plates by the cell being diluted to certain density, makes every hole only have a Growth of Cells.From the hole forming cell colony, get supernatant do enzyme-linked immunosorbent assay (ELISA), qualification positive colony.Can repeated cloning several times, until the positive porosity of hybridoma reaches 100%.Hybridoma enlarged culturing after cloning is done Identification of the antibodies;
(6) induction of mouse ascites: inoculating first 10 days of hybridoma, to mouse peritoneal injecting fluid paraffin 0.5mL/ only, then inoculates positive hybridoma cell 1.0 × 10
6/ only, and centrifugal after within 10 days, collecting ascites afterwards, measure antibody titer, and monoclonal antibody purification;
(7) purifying of monoclonal antibody: the monoclonal antibody in Affi-Gel protein A-agarose affinity column (Bio-Rad) purifying ascites supernatant.
When obtaining described hybridoma, can Zooblast cultivation method conveniently, the hybridoma described in cultured and amplified in vitro, thus the monoclonal antibody described in making it to secrete.As a kind of embodiment, described monoclonal antibody can be prepared by following preparation method: (1) provides adjuvant pretreated mouse; (2) in mouse peritoneal, described hybridoma is inoculated and secrete monoclonal antibody; (3) extract ascites, be separated the monoclonal antibody described in obtaining.As a kind of mode, the method being separated monoclonal antibody from ascites is: collect ascites, with through ammonium sulfate, sad precipitation, then pre-installs column chromatography with Protein G, obtains highly purified anti-ASGPR monoclonal antibody.
Monoclonal antibody of the present invention can also utilize recombination method prepare or utilize Peptide synthesizer to synthesize.Those skilled in the art all understand, and are obtaining the hybridoma cell line of described monoclonal antibody or after learning described monoclonal antibody by means such as order-checkings, those skilled in the art can obtain described antibody easily.
Monoclonal antibody of the present invention is for the large subunit of ASGPR H1, because the large subunit of H1 plays a significant role in recognition ligand and mediation endocytosis, its expression amount is also 3 times of H2 small subunit, and the antibody therefore for H1 subunit is more valuable.
The present invention also provides the hybridoma cell strain that can produce said monoclonal antibody, and it is preserved in China typical culture collection center (China, Wuhan, Wuhan University), and preserving number is: CCTCC NO:C201370.
Detection kit
After obtaining anti-ASGPR monoclonal antibody of the present invention, those skilled in the art detect ASGPR albumen and concentration thereof in sample delicately by number of ways, and the technology of employing can be technology conventional in field of immunology.Comprise qualitative detection and quantitative detecting method.Preferably, described qualitative detection comprises: there is situation by the method identifier asialoglycoprotein receptor of western blotting method or immunofluorescence; Preferably, described detection by quantitative comprises: carried out quantitatively the intensity of cell expressing people asialoglycoprotein receptor or positive rate by flow cytometry, or is carried out quantitatively the expression of people's asialoglycoprotein receptor by double antibody sandwich ELISA.
The invention provides a kind of for detecting the detection kit that whether there is ASGPR in sample, containing anti-ASGPR monoclonal antibody of the present invention in this test kit.
Except containing except anti-ASGPR monoclonal antibody of the present invention in described test kit, other detection reagent or assistant agent can also be comprised, as developer, marker, two anti-, sensitizer etc.Those skilled in the art should be understood that the detection kit of various version is all included in the present invention, as long as wherein make use of anti-ASGPR monoclonal antibody of the present invention as the reagent with ASGPR specific binding.
As used herein, described " marker " refers to that whether and the mark of the amount existed existence for determining ASGPR in detected sample, and it also can directly be coupled in anti-ASGPR monoclonal antibody, forms immune conjugate.Determining coated antibody that test kit of the present invention adopts and/or after detecting antibody, can adopt this area conventional for the various markers detecting antibodies and carry out detecting.The present invention has no particular limits adopted marker, as long as can with described detection antibodies, and can indicate exactly after appropriate processing the existence of ASGPR albumen in detected sample whether and the marker of amount be all available.Described marker directly can be arranged at and detect on antibody; Or described marker also can be arranged at two of the anti-detection antibody of specificity and resist.Those skilled in the art according to the kind of adopted antibody and characteristic, can select suitable marker.Such as, described marker can be selected from: horseradish peroxidase (HRP), alkaline phosphatase (AP), vitamin H, fluorescein, glucose oxidase, beta-D-galactosidase, urase, catalase or glucoamylase.
When adopting some enzyme marker as implied above, also need the substrate adopting some to be combined with corresponding enzyme, thus carry out reporter marker thing by modes such as colour developings there is situation or amount.As used herein, described " substrate " refers to and can be labeled thing institute catalyzed coloration, for showing the recognition signal detecting antibody and ASGPR and occur to combine.Described substrate is such as: for O-Phenylene Diamine (OPD), tetramethyl benzidine (TMB), the ABTS of horseradish peroxidase; For the p-nitrophenyl phosphoric acid ester (p-nitrophenyl phosphate, p-NPP) of alkaline phosphatase; Etc..Those skilled in the art according to the kind of adopted marker and characteristic, can select suitable substrate.
In order to obtain quantitative result, the standard substance of the multiple ASGPR albumen containing concentration known can be set in testing process.Method to set up for standard substance can adopt conventional method.
In addition, in described test kit, also working instructions can be comprised, for illustration of the using method of the reagent wherein loaded.
In another preference of the present invention, above-mentioned monoclonal antibody can also with mark coupling, described mark is selected from: horseradish peroxidase, alkaline phosphatase, vitamin H or fluorescein.
The present invention also provides a kind of separation method of tumour cell.
The application of monoclonal anti body sorting liver cancer cell of the present invention
Present invention also offers a kind of separation method of the liver cancer cell that circulates, comprise the steps: that (a) is separated mononuclearcell (wherein containing circulation liver cancer cell) from the vitro samples containing circulation liver cancer cell; B () mixes with mononuclearcell with first antibody, thus form " liver cancer cell-first antibody " binary complex; Described first antibody is described monoclonal antibody; C () has the magnetic bead of second antibody to mix with described binary complex with coupling, thus form " liver cancer cell-first antibody-second antibody-magnetic bead " tetraplex; (d) tetraplex in magnetic separation step (c).
Antibody of the present invention is monoclonal antibody, and specificity is higher than polyclonal antibody.Further, antibody of the present invention is the mouse source antibody for human antigen, and the technology of preparing of mouse monoclonal antibody is ripe, simple to operate, can produce in a large number rapidly; Can match with two of current commercially available marked by magnetic bead anti-(for mouse source).
Antibody of the present invention is mouse IgG 1.Two of current marked by magnetic bead on the market resists and comprises anti-mouse IgG1 and IgG two kinds (comprising IgG1, IgG2a, IgG2b and IgG3), and obvious specificity better anti-igg 1 magnetic bead is better; Although also have antibiotin magnetic bead on the market, use this magnetic bead also to need prior applicating biotin traget antibody, add operation steps, not as directly selecting two diamagnetic pearls.
Monoclonal antibody recognition site of the present invention is positioned at ASGPR extracellular fragment.Permeable membrane is needed to operate when the antibody of application identification ASGPR born of the same parents inner segment or ASGPR total length carries out marked by magnetic bead, but permeable membrane operation produces damage to circulation liver cancer cell, one causes circulation liver cancer cell to be intactly separated, even if two have been separated and also cannot carry out follow-up research or cultivation.And monoclonal antibody of the present invention overcomes this technical problem, achieve the technique effect without the need to carrying out permeable membrane operation.
Therefore, mouse IgG 1 monoclonal antibody that application ASGPR H1 large subunit extracellular fragment 61-291 aminoacid sequence (SEQ ID NO:1) is prepared for immunogen, compared with known ASGPR antibody, has excellent technique effect.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, conveniently condition such as J. Pehanorm Brooker etc. is write usually, Molecular Cloning: A Laboratory guide, the third edition, Science Press, the condition described in 2002, or according to the condition that manufacturer advises.
The preparation of embodiment 1, monoclonal antibody
ASGPR cDNA (encoding amino acid sequence is as the ASGPR fragment of SEQ ID NO:1) is obtained by RT-PCR amplification, with pGEX-4T-1 plasmid (purchased from American Invitrogen company) for carrier, in the BamH I being inserted into this carrier and EcoR I site, build and add that the ASGPR of GST label expresses recombinant vectors, Transformed E .coli BL21 competent cell, through screening and cultivation, isopropylthiogalactoside (IPTG) abduction delivering, GST affinity column purifying, 12%SDS-PAGE detection fusion protein expression.
SQNSQLQEELRGLRETFSNFTASTEAQVKGLSTQGGNVGRKMKSLESQLEKQQKDLSEDHSSLLLHVKQFVSDLRSLSCQMAALQGNGSERTCCPVNWVEHERSCYWFSRSGKAWADADNYCRLEDAHLVVVTSWEEQKFVQHHIGPVNTWMGLHDQNGPWKWVDGTDYETGFKNWRPEQPDDWYGHGLGGGEDCAHFTDDGRWNDDVCQRPYRWVCETELDKASQEPPLL(SEQ ID NO:1)
The methods such as the production of mouse immune, cytogamy, positive hybridoma cell colony screening, ascites and antibody purification all traditionally merge hybridoma technology and carry out.
Through the separation screening to a large amount of hybridoma, finishing screen chooses the excellent hybridoma cell strain hASGPR-021 of a strain, the anti-ASGPR monoclonal antibody that its secretion specificity is good.
The detection of embodiment 2, monoclonal antibody
1. the qualification of monoclonal antibody subclass
Carry out according to monoclonal antibody subgroup identification test kit specification sheets.
As a result, recording monoclonal antibody of the present invention is IgG1 hypotype.
②ELISA
ELISA method routinely.
3. immunoblotting (Western blot)
Western blotting method routinely.
4. flow cytometry (flow cytometry, FACS)
Prepare human liver cell system WRL68 and Bel7402 Hep3B, Huh7, HepG2, PLC/PRF/5 (above clone is all purchased from Chinese Academy of Sciences's Shanghai cell bank).The ASGPR expression of part clone has bibliographical information [Park JH, Cho EW, Shin SY, Lee YJ, Kim KL.Detection ofthe asialoglycoprotein receptor on cell lines of extrahepatic origin.BiochemBiophys Res Commun, 1998,244:304-311].
The above-mentioned clone of cellar culture, cell 2 in vegetative period hole of taking the logarithm respectively, trysinization, the centrifugal 5min of 1500rpm, respectively collects 1 × 10
6individual cell, be placed in respectively and be labeled as 1, in the centrifuge tube of 2, wherein 1 is blank pipe (Control), 2 is anti-ASGPR monoclonal antibody detector tube (ASGPR), PBS washes twice, abandon supernatant, the anti-ASGPR monoclonal antibody 100 μ L of PBS dilution is added in No. 2 pipes, the PBS of identical amount is added in No. 1 pipe, all hatch 1h at 37 DEG C, PBS washes 2 times, the centrifugal 5min of each 1500rpm, abandon supernatant, No. 2 pipes add the anti-100 μ L of FITC mark goat anti-mouse IgG two of PBS dilution, No. 1 pipe adds the PBS of equivalent, normal temperature lucifuge hatches 45min, PBS washes 2 times, the centrifugal 5min of each 1500rpm, abandon supernatant, often manage and add 200 μ L PBS, upper machine, data collection and analysis.
Result is as Fig. 1, and different hepatoma cell line ASGPR expresses and there are differences, and wherein HepG2 cell expressing rate is the highest, and detected result conforms to bibliographical information.Illustrate that anti-ASGPR monoclonal antibody of the present invention can be applicable to accurately detect the expression of ASGPR in clone.
5. immunohistochemical staining
Normal people's hepatic tissue, liver cancer tissue, hepatic metastasis of colonic carcinoma tissue paraffin section de is through dimethylbenzene and graded ethanol dewaxing aquation, structure same regimen acid salt damping fluid high temperature antigen retrieval, often open section dropping intrinsic oversxidase blocker, normal temperature hatches 10min, PBS washes, goat NIS is closed, normal temperature hatches 30min, drip the anti-ASGPR monoclonal antibody of PBS appropriateness dilution, 4 DEG C of overnight incubation, PBS washes, drip toughener normal temperature and hatch 20min, PBS washes, drip HRP ELIAS secondary antibody, hatch 30min for 37 DEG C, PBS washes, DAB develops the color 5min, tap water, Hematorylin is redyed, PBS is anti-blue, gradient alcohol dehydration is dry, dimethylbenzene is transparent, dry, neutral gum mounting, microscopic examination is taken pictures.
Result is as Fig. 2, and visible ASGPR specificity expression is hepatic parenchymal cells surface in normal liver tissue, liver cancer tissue and secondary liver cancer cancer beside organism, and does not express in mesenchymal cell and intestinal cancer metastasis cell.
6. immunohistofluorescence stain
The dewaxing of liver cancer tissue section dimethylbenzene, graded ethanol aquation, structure same regimen acid salt damping fluid high temperature antigen retrieval, endogenous peroxydase blocker normal temperature hatches 10min, PBS washes 3min × 3 time, goat NIS is closed, normal temperature hatches 30min, drip the anti-ASGPR monoclonal antibody (two anti-contrasts add the PBS of equivalent) of PBS appropriateness dilution, 4 DEG C of overnight incubation, PBS washes 3min × 3 time, drip cy3 mark goat anti-mouse IgG two to resist, 37 DEG C of lucifuges hatch 30min, PBS washes 3min × 3 time, DAPI contaminates core 3min, PBS washes 3min × 3 time, natural air drying, anti-fluorescence quenching mounting, fluorescence microscopy Microscopic observation, gather picture.
Result is as Fig. 3, and visible anti-ASGPR monoclonal antibody of the present invention can dye and express the hepatoma cell membrane of ASGPR, has good specificity.
7. immunocytochemical stain
Cellar culture hepatocellular carcinoma H22 and breast cancer cell MCF-7, at logarithmic phase collecting cell, choose hepatoma cell line to mix with breast cancer cell line equal proportion, be planted in same hole and carry out cell climbing sheet, overnight incubation, creep plate was taken out in second day, PBS rinses, 4% formaldehyde is fixed, tap water, 0.4% Triton permeable membrane, 3% endogenous peroxydase blocker blocks, goat NIS is closed, drip the anti-ASGPR monoclonal antibody of PBS appropriateness dilution, 4 DEG C of overnight incubation, PBS washes, drip toughener normal temperature and hatch 20min, PBS washes, drip HRP ELIAS secondary antibody, hatch 30min for 37 DEG C, PBS washes, DAB develops the color 5min, tap water, Hematorylin is redyed, PBS is anti-blue, gradient alcohol dehydration is dry, dimethylbenzene is transparent, dry, neutral gum mounting, microscopic examination is taken pictures.
Result is as Fig. 4, visible anti-ASGPR monoclonal antibody can express the cytolemma of HepG2 cell (karyon is less) of ASGPR by specific stain, then specific binding does not occur for the breast cancer cell MCF-7 (karyon is larger) not expressing ASGPR.
8. immunocyte fluorescent dye
Cellar culture hepatocellular carcinoma H22 and breast cancer cell MCF-7, at logarithmic phase collecting cell, choose hepatoma cell line to mix with other tumor cell line equal proportions, be planted in same hole and carry out cell climbing sheet, overnight incubation, creep plate was taken out in second day, PBS rinses, 4% formaldehyde is fixed, tap water, 0.4% Triton permeable membrane, goat NIS is closed, drip the anti-ASGPR monoclonal antibody of PBS appropriateness dilution, 4 DEG C of overnight incubation, PBS washes 3min × 3 time, drip cy3 mark goat anti-mouse IgG two to resist, 37 DEG C of lucifuges hatch 30min, PBS washes 3min × 3 time, DAPI contaminates core 3min, PBS washes 3min × 3 time, natural air drying, anti-fluorescence quenching mounting, fluorescence microscopy Microscopic observation, gather picture.
Result is as Fig. 5, and visible anti-ASGPR monoclonal antibody can express the cytolemma of HepG2 cell of ASGPR by specific stain, then specific binding does not occur for the breast cancer cell MCF-7 not expressing ASGPR.
Embodiment 3, utilize monoclonal antibody separation andpreconcentration circulation liver cancer cell
Main raw: 5mL liver cancer patient volunteer peripheral blood, EDTA anti-freezing.Ficoll-Paque Plus, purchased from GE Healthcare.Mouse anti human ASGPR monoclonal antibody (IgG1) of the present invention.Magnetic separator, anti-Mouse IgG1MicroBeads, MS magnetic sorting post, 30 μm of filter screens, CK3-6H5 (antibody for epithelium source tumour cell) are all purchased from German Miltenyi company.
400R desk centrifuge (band cell centrifugation smear annex), purchased from German Heraeus company.Poly-lysine slide, steps new company purchased from Fujian.SP two step method immunohistochemical kit, steps Newbiotics, Inc. purchased from Foochow.PBS washing lotion (containing 2mM EDTA and 0.5%BSA) autogamy.
Step:
(A) Ficoll separating peripheral blood mononuclear cells
1) 5mL EDTA anticoagulation and 5mL PBS washing lotion are mixed in 50mL centrifuge tube, be laid on 7mL Ficoll, 18 DEG C, 600g, centrifugal 25min.
2) upper serum is abandoned in suction.
3) draw mononuclearcell layer, be placed in 15mL centrifuge tube.
4) 3 times of volume PBS washing lotions are added, 18 DEG C, 300g, centrifugal 10min.
5) supernatant is abandoned in suction, and 8mL PBS washing lotion is resuspended, 18 DEG C, 300g, centrifugal 10min.
6) thoroughly supernatant is abandoned in suction.
(B) Beads enrichment circulation liver cancer cell
1) 130 μ L PBS washing lotion re-suspended cells.
2) 20 μ L mouse anti human ASGPR monoclonal antibodies are added, mixing.
3) incubated at room 30min.
4) add 5mL PBS washing lotion, 18 DEG C, 300g, centrifugal 10min, inhale and abandon supernatant.
5) previous step is repeated.
6) 80 μ L PBS washing lotions are added.
7) 20 μ L anti-Mouse IgG1MicroBeads are added, mixing.
8) 4 DEG C, 15min is hatched.
9) 5mL PBS washing lotion is added, 18 DEG C, 300g, centrifugal 10min.
10) supernatant is abandoned in suction, 500 μ L PBS washing lotions resuspended (attention tries not to produce bubble).
11) MS magnetic sorting post and filter screen are installed.
12) 500 μ L PBS washing lotions add filter screen.
13) below treating sorting post, drop just stops, and namely adds 500 μ L cell suspensions to filter screen.
14) 500 μ L PBS washing lotions are added in 15mL centrifuge tube, wash residual cell.
15) when sorting post upper liquid has flowed soon, 500 μ L cell suspensions are added.
16) when sorting post upper liquid has flowed soon, 500 μ L PBS washing lotions are added.
17) previous step 2 times are repeated.
18) below treating sorting post, drop just stops, and sorting post is shifted out magnetic field, is placed on another aseptic 15mL centrifuge tube.
19) add 1mL PBS washing lotion in sorting post, with piston, liquid is pushed in centrifuge tube fast immediately.
20) previous step is repeated.
21) 18 DEG C, 300g, centrifugal 10min.
22) supernatant is abandoned in suction, and 100 μ L PBS washing lotions are resuspended.
(C) the immunofluorescence dyeing qualification of circulation liver cancer cell
Collect above-mentioned cell suspension and carry out cell smear, dry for 52 DEG C, fix through 4% formaldehyde, 0.4% Triton permeable membrane, 3% endogenous peroxydase blocker blocks, goat NIS is closed, application liver cell specificity Hep Par1 antibody or epithelial cell specific C K antibody, dye in conjunction with hemocyte specific C D45 antibody, 4 DEG C of overnight incubation, PBS washes, drip cy3 anti-mouse two anti-anti-with Alexa fluor488 Chinese People's Anti-Japanese Military and Political College mouse two, hatch 30min for 37 DEG C, PBS washes, DAPI contaminates core, PBS washes, naturally dry, anti-quenching of fluorescence mountant mounting, microscopic examination is taken pictures.
Result:
Result as shown in Figure 6.Liver cancer cell presents the Hep Par1 positive, DAPI is positive, CD45 is negative, and cell is in circle to oval, and nucleus is complete, has the malignant cell features such as cell volume is large, karyoplasmic ratio is high.
Biomaterial preservation
In the application, hybridoma cell strain hASGPR-021 has been preserved in China typical culture collection center (China, Wuhan, Wuhan University), and preserving number is: CCTCC NO:C201370, and preservation day is: on May 24th, 2013.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. a monoclonal antibody, it is the monoclonal antibody that the hybridoma cell strain being CCTCC NO:C201370 by preserving number is secreted.
2. monoclonal antibody as claimed in claim 1, it is characterized in that, described monoclonal antibody is Immunoglobulin Isotype IgG1 type.
3. a hybridoma cell strain, it is CCTCCNO:C201370 at the preserving number of China typical culture collection center.
4. an immune conjugate, is characterized in that, described immune conjugate comprises: monoclonal antibody according to claim 1 and the marker be attached thereto; Described marker is selected from: horseradish peroxidase, alkaline phosphatase, vitamin H or fluorescein.
5. a detection kit, is characterized in that, comprising:
Monoclonal antibody according to claim 1; Or
Hybridoma cell strain according to claim 3; Or
Immune conjugate according to claim 4.
6. test kit as claimed in claim 5, is characterized in that, wherein also comprise:
Second antibody, described second antibody specific recognition monoclonal antibody according to claim 1;
Immunomagnetic beads;
Developer;
Washings; And/or
Stop buffer.
7. the purposes of monoclonal antibody according to claim 1, for the preparation of detection reagent or the test kit of specific detection people asialoglycoprotein receptor.
8. purposes as claimed in claim 7, it is characterized in that, described detection people asialoglycoprotein receptor comprises: (1) qualitative detection people asialoglycoprotein receptor; Or (2) detection by quantitative people asialoglycoprotein receptor.
9. purposes as claimed in claim 7, is characterized in that, described detection reagent or test kit also for:
The discriminating of hepatocellular carcinoma and secondary liver cancer;
The separation and detection of circulation liver cancer cell;
Liver imaging; With
The targeted delivery of liver drug or gene.
10. circulate the separation method of liver cancer cell, it is characterized in that, comprise the steps:
A () is separated mononuclearcell from the vitro samples containing circulation liver cancer cell;
B () mixes with mononuclearcell with first antibody, thus form " liver cancer cell-first antibody " binary complex; Described first antibody is monoclonal antibody according to claim 1;
C () has the magnetic bead of second antibody to mix with described binary complex with coupling, thus form " liver cancer cell-first antibody-second antibody-magnetic bead " tetraplex; With
Tetraplex in (d) magnetic separation step (c);
Preferably, the separation method that described separation mononuclearcell is taked is density gradient cytopheresis;
Preferably, described sample is selected from marrow, blood or other body fluid; Preferably, other body fluid described are lymph liquid, hydrothorax or ascites.
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| CN113508141B (en) * | 2019-02-01 | 2024-10-29 | 住友化学株式会社 | Antibodies and functional fragments thereof |
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| CN113917162A (en) * | 2021-12-14 | 2022-01-11 | 江苏为真生物医药技术股份有限公司 | Application of asialoglycoprotein receptor fragment sH2a as marker |
| CN114805545A (en) * | 2021-12-28 | 2022-07-29 | 苏州和锐生物科技有限公司 | Capture antigen for detecting anti-asialoglycoprotein receptor antibody and detection method |
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